Oceanological and Hydrobiological Studies

International Journal of Oceanography and Hydrobiology

Volume 44, Issue 1, March 2015 ISSN 1730-413X pages (68-73) eISSN 1897-3191

Contracaecum spp. from endemic Baikal : the Baikal yellowfin grewingkii (Dybowski, 1874) and the longfin Baikal Cottocomephorus inermis (Yakovlev, 1890) by Abstract

1 Olga Rusinek All the nematodes found in body cavities of the examined 2 endemic Baikal fishes: 88 Baikal yellowfinCottocomephorus Marek Kulikowski grewingkii (Dybowski, 1874) and 35 longfin Baikal sculpin Katarzyna Najda2 Cottocomephorus inermis (Yakovlev, 1890) were identified as 2,* Contracaecum osculatum baicalensis (Mozgovoi and Ryjikov, Jerzy Rokicki 1950) L3 larvae. The prevalence, mean intensity, intensity range and abundance of the nematodes in C. grewingkii were 37.5%, 2.55, 1-31, and 0.96, respectively, the corresponding values in C. inermis were 60.0%, 2.43, 1-10, and 1.46. The infestation level in C. grewingkii was significantly higher than DOI: 10.1515/ohs-2015-0007 in C. inermis (Mann-Whitney U-test, p<0.02). The number of parasites was found to increase with the length. Although Category: Original research paper in both and C. inermis, the anisakids were more frequent Received: September 29, 2014 in males (prevalence of 52.17 and 67.76%, respectively) than Accepted: November 19, 2014 in females (prevalence of 35.39 and 42.86%, respectively), differences between the sexes in the infestation level in the two were not significant (Mann-Whitney U-test, P= 0.09 and P=0.23, respectively). The molecular method applied (PCF-RFLP) allows to identify all the nematodes in 1 Baikal Museum of Irkutsk Scientific Center both examined fish species asC. osculatum baicalensis. of Siberian Branch of the Russian Academy of Sciences, Akademicheskaya str.,1, 664520 Listvyanka, Russia

2Department of Invertebrate Zoology and Parasitology, University of Gdańsk, ul. Wita Stwosza 59, 80-308 Gdańsk, Poland

Key words: Contracaecum osculatum baicalensis, Anisakidae, Cottocomephorus, PCR - RFLP

* Corresponding author: [email protected]

The Oceanological and Hydrobiological Studies is online at oandhs.ocean.ug.edu.pl

©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 69 Contracaecum osculatum baicalensis from Baikal Cottocomephorus spp.

Mattiucci & Nascetti 2008). Molecular data show C. Introduction osculatum baicalensis to be genetically different from the remaining five C. osculatum sibling species and The parasitic nematode Contracaecum osculatum validate its identification as a true biological species baicalensis (Mozgovoi and Ryjikov 1950) of the within the C. osculatum complex (D’Amelio et al. family Anisakidae is a common endemic species 1995). At the larval stage, C. osculatum baicalensis in (Rusinek 2007). The nematode’s was recognized by allozymes in the Baikal endemic complex life cycle involves at least two intermediate fish species: C. grewingkii, C. inermis, hosts (Sudarikov & Ryzhikov 1951). Hypothetically, baicalensis, lavaretus, C. autumnalis some invertebrate organisms: the dominant Baikal migratorius and Thymallus arcticus, which represent zooplankter Macrohectopus branickii (Dybowsky prey items of Pusa sibirica in Lake Baikal (Mattiucci 1874) (, Gammaridae) and harpacticoid & Nascetti 2008). copepods may serve as the first intermediate hosts We hypothesize that new information regarding (Bauer 1987). It is reasonable to assume that the first the Baikal Cottocomephorus spp. parasites is essential intermediate hosts occur in abundance (Rusinek for exploring changes in the infection level and 2007). The second intermediate hosts include fish for refining the identification techniques. This species endemic to Lake Baikal: the benthopelagic information will allow us to identify the distribution Baikal Cottocomephorus grewingkii (Dybowski 1874) of intermediate hosts in Lake Baikal and to arrive at and the longfin Baikal sculpin, Cottocomephorus a better understanding of host-parasite interactions. inermis (Yakovlev 1890) (, Cottoidei), which support the parasites as L3 larvae in their coelom (Lâjman 1933, Dogel’ et al. 1949, Materials and methods Sudarikov & Ryzhikov 1951, Zaika 1965, Rusinek et al. 2007). In addition, many other fish species Parasites (salmonids, coregonids, thymallids, and lotids) can act as the second intermediate hosts of C. The material for this study was collected from 88 osculatum baicalensis (Zaika 1965, Rusinek 2007). and 35 individuals of C. grewingkii and C. inermis, The nematode reaches the maturity in the stomach respectively, caught in July-September 2012 near and intestine of the Pusa sibirica (Gmelin Listvyanka, on the southern shore of Lake Baikal 1788) which feeds on the fish species mentioned (51°51’ 05.00”N; 104° 51’ 55.00”E). Each fish was above. The Baikal seal is the only known definitive sexed and measured (lt) to the nearest 1 mm. The host of the parasite (Sudarikov & Ryzhikov 1951, C. grewingkii specimens were divided into 3 length Rusinek 2007). classes, while C. inermis into 2 classes – on account As in other anisakid species complexes e.g. of their length distribution – so that the number of Anisakis simplex (Mattiucci et al. 1997), speciation fish in each interval was more than 15. Nematodes within the C. osculatum complex appears to be were isolated from host body cavities, rinsed in accompanied by poor morphological differentiation, water, and stored in 70% ethanol until further and morphological identification of sibling species is analysis. Subsequently, each parasite was dissected unreliable at both larval and adult stages. In contrast, into 3 parts. Semi-permanent mounts were prepared nematode identification at any developmental stage is of the anterior and posterior part of each specimen possible by molecular methods (Kijewska et al. 2002), (Rolbiecki 2002) and examined under a compound which is important for the study of the parasites’ life light microscope. The nematodes were identified cycles, biology, , and pathology they may using the identification key (Bauer 1987). The middle cause. These anisakid taxa can be characterized part of each individual was fixed in 70% ethanol and using several different molecular markers, including used for molecular identification. allozymes, nuclear ribosomal DNA (rDNA), internal transcribed spacer regions (ITS), single strand DNA isolation and amplification conformation polymorphism mitochondrial DNA (mtDNA), and cytochrome oxidase 2 (cox2) sequence The morphological identification of nematodes analyses (Zhu et al. 1998, Mattiucci & Nascetti 2007, was checked on randomly selected specimens

©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. 70 Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 Olga Rusinek, Marek Kulikowski, Katarzyna Najda, Jerzy Rokicki digested by endonucleases, using a molecular (1982). All the statistical analyses were performed key based upon DNA patterns of the ITS1-5.8S- with STATISTICA 12.0. Non-parametric tests ITS2 fragment (Zhu et al. 2000, Dzido 2011). The (Mann-Whitney U-test, and Spearman’s rank nematode DNA was isolated as described by Hoarau correlation) were used because the distribution of et al. (2002). Amplification of an rDNA fragment was parasites deviated from the normal distribution due accomplished according to Zhu et al. (1998). Each to the accumulation of larvae and diet preferences of reaction mixture (total volume of 16 µl) contained the fish. The two fish species were compared in terms 1 µl of isolated genomic DNA, 1 U DyNAzyme II of their nematode infection level. The frequency DNA Polymerase (Finnzymes, Vantaa, Finland), distributions of parasite-fish sex correlation was dNTPs (250 µM each), 100 µM of each primer [NC2 examined as well. In addition, the data were tested and NC5 (Zhu et al. 1998)] and buffer 1 × (10 mM for significance of differences in the load of parasites Tris-HCl (pH = 8.4), 50 mM KCl, 0.1% Triton X-100, between the length classes. and 1.5 mM MgCl2). Amplification was carried out in a Techne Progene (Stone, U.K.) thermocycler Results as follows: initial denaturation for 5 min. at 94°C, followed by 30 cycles for 30 sec. at 94°C, annealing Morphological identification resulted in assigning at 60°C for 30 sec., and extension at 72°C for 30 sec., all the nematodes isolated from the yellowfin and terminated by the extension cycle at 72°C for 5 min. longfin Baikal sculpin to Contracaecum osculatum PCR products were separated electrophoretically baicalensis (Mozgovoi & Ryjikov 1950) L3 larvae. on 1% agarose gels and visualized by staining with In C. grewingkii, the infestation prevalence, mean ethidium bromide. intensity, intensity range, and nematode abundance Restriction fragment length polymorphism were 37.5%, 2.55, 1-31, and 0.96, respectively The amplified DNA samples were digested with (Table 1). When assessed for the yellowfin sex- TaqI, RsaI, XbaI, and BsuRI (Fermentas, Vilnius, related distribution (65 females and 23 males), the Lithuania) restriction enzymes and the products anisakids proved to occur more frequently in males were separated electrophoretically on 4% agarose gels than in females (prevalence of 52.17 and 35.39%, and visualized by staining with ethidium bromide respectively), but they were more numerous females (Sambrook 1989). The pUC Mix Marker (0.5 µg µl- (mean intensity of 2.74) than in males (1.75). 1; Marker 8, Fermentas) was used as a marker. The However, the Mann-Whitney U-test showed that product sizes were compared to the rDNA ITS1- the differences between the sexes in the C. grewingkii 5.8S-ITS2 digestion pattern key (Zhu et al. 2000, infestation level were not significant (P=0.09). The Dzido 2011). number of parasites in C. grewingkii was found to increase significantly with fish length (Spearman’s Statistical treatment rank correlation coefficient rs=0.279; P<0.009) (Table 1). In C. inermis, the prevalence of infestation, mean The prevalence, intensity, mean intensity, and intensity, intensity range, and parasite abundance abundance of the parasitic infection for all detected were 60.0%, 2.43, 1-10, and 1.46, respectively. The parasites were calculated according to Margolis et al. females (7 specimens) were less infected than males

Table 1 Prevalence, mean intensity and abundance of L3 C. osculatum baicalensis larvae in C. grewingkii length classes SD - standard deviation lt (mm) <100 100-110 >110 All Number of fish 25 34 29 88 Prevalence (%) 28.00 35.29 48.28 37.50 Mean intensity ± SD 1.71 ± 1.11 1.33 ± 0.65 4.00 ± 7.87 2.55 ± 5.21 Abundance ± SD 0.48 ± 0.96 0.47 ± 0.75 1.93 ± 5.74 0.96 ± 3.40

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©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 71 Contracaecum osculatum baicalensis from Baikal Cottocomephorus spp.

(28 specimens), the corresponding mean intensity data being 38.5, 1.5, and 1-16. On the other hand, values were 1.33 vs. 2.53. However, the sex-related the infestation data found in this study were higher difference in the infestation level proved non- than those reported by Sudarikov & Ryzhikov (1951) significant (Mann-Whitney U-test, P=0.23). The who found prevalence and mean intensity to be largest fishes were observed to host the highest 19.4% and 1-2, respectively. Those authors found the numbers of nematodes (Spearman’s rank correlation fish caught in the vicinity of the Baikal seal colony coefficient rs=0.641; P<0.00005) (Table 2). to be particularly highly affected by the parasite A total of 88 C. grewingkii yielded 84 nematodes, infestation. They assigned this finding to a high 51 nematodes were isolated from 35 C. inermis. The probability of planktonic crustaceans occurring close interspecific difference in the number of parasites to the seal colonies eating nematode eggs released proved significant (Mann-Whitney U-test; P<0.02). from the seal feces. Those crustaceans are then The PCR-RFLP technique was used to examine consumed by planktivorous fish which are preyed the reliability of morphological identification. The upon by the Baikal seals, which closes the life cycle restriction patterns for identification of sibling species of C. osculatum baicalensis. However, the analysis in C. osculatum s.l. were specific to C. osculatum of data on the infestation level of C. grewingkii near baicalensis (XbaI: 960 bp; RsaI: 400, 270 bp; BsuRI: Listvyanka shows that this theory does not hold 420, 260 and TaqI: 210, 180, 170, 140, 90 bp) (Zhu entirely, because Zaika (1965) did not find any et al. 2000, Dzido 2011). Thus, all the nematode anisakids in the yellowfin Baikal sculpin in that specimens isolated from the two studied fish species area. Therefore, it is necessary to consider additional proved to represent C. osculatum baicalensis. aspects, such as the fact that seals leave their colony

Table 2 Prevalence, mean intensity and abundance of C. osculatum baicalensis L3 larvae in C. inermis length classes SD - standard deviation lt (mm) ≤120 >120 All Number of fish 16 19 35 Prevalence (%) 43.75 73.68 60.00 Mean intensity ± SD 1.14 ± 0.35 3.07 ± 2.98 2.43 ± 2.54 Abundance ± SD 0.50 ± 0.62 2.26 ± 2.94 1.46 ± 2.29

on account of migrations and hunting. There is also Discussion a possibility that, because of the size and the depth of Lake Baikal and the associated variability in the lake’s The presence of C. osculatum baicalensis L3 current intensity and direction, nematode eggs and larvae in the Baikal C. grewingkii had already been planktonic crustaceans, the first intermediate hosts, reported by other authors (Lâjman 1933, Dogel’ et are being spread throughout the lake. It should be al. 1949, Sudarikov & Ryzhikov 1951, Zaika 1965, noted, however, that the parameters of C. grewingkii Rusinek 2007, Mattiucci & Nascetti 2008). All those infestation with C. osculatum baicalensis in other studies resulted in different infestation parameters Baikal areas differed from those in Listvyanka. which were dependent on the fish collection site Sudarikov & Ryzhikov (1951) reported prevalence and season. A comparison of literature data on the and mean intensity off the village Goloustnoe to be C. osculatum baicalensis infestation of the yellowfin 53.1% and 5, respectively, the corresponding data for Baikal sculpin from Listvyanka with the results of the Barguzinskiy Zaliv being 37.5% and 5-6 r. Similar this work showed a similarity with data reported by differences were observed by Zaika (1965) who, e.g. Rusinek (2007): the prevalence, mean intensity, however, provided no information on the infestation and intensity range were as follows in this study: and fish collection site location. 37.5, 2.55, 1-31, respectively, the corresponding The anisakid fauna ofC. inermis was studied

©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. 72 Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 Olga Rusinek, Marek Kulikowski, Katarzyna Najda, Jerzy Rokicki by Rusinek (2007), Zaika (1965) and Mattiucci nematodes were collected from two species of the & Nascetti 2008. The longfin Baikal sculpin from Baikal fish. Application of molecular techniques Listvyanka, examined in the present study, showed results in a more reliable identification of parasite the infestation level similar to that reported by species. Differences in the number and the presence Rusinek (2007): 60.0% vs. 53.3%, 2.43 vs. 1.07, and of restriction sites for endonucleases used allowed 1-10 vs. 1-7 being the prevalence, mean intensity, the differentiation between C. osculatum baicalensis and intensity range values, respectively. On the and another C. osculatum species. other hand, the C. inermis infestation level found in this study was lower than that reported from other Acknowledgements Baikal areas, e.g. Nizhne-Angarsk (prevalence 80% and intensity range 1-3) and Maloe More (Zaika This study was partially supported by the 1965). By analogy to C. grewingkii, it can be assumed National Science Centre grant No. DEC-2011/01/B/ that the infection parameters change due to similar NZ8/04194 and by the European Social Fund as part processes (Sudarikov & Ryzhikov 1951). of the project “Educators for the elite − integrated Macrohectopus branickii at all its developmental training program for PhD students, post-docs and stages forms the major component of the Baikal professors as academic teachers at the University of zooplankton (Kozhov 1963, Rudstam et al. 1992, Gdansk” within the framework of the Human Capital Melnik et al. 1995) and significantly contributes Operational Programme, Action IV. to the diets of both C. grewingkii and C. inermis (Zubin 1992, Dzyuba et al. 2000, Dzyuba 2004). The amphipod has been found to serve as a host References of C. osculatum baicalensis (Bauer 1987), but other zooplankters could act as the first intermediate Bauer, O.N. (1987). Key to the parasites of freshwater fish of the hosts as well. The Baikal zooplankton includes two USSR. Metazoan parasites. Leningrad: Izdatel’stvo Nauka (in copepod species: Epischura baicalensis and Cyclops Russian). kolensis (Kozhov 1963, Mazepova 1998), high D’Amelio, S., Mattiucci, S., Paggi, L., Koie, L., Podvyaznaya, quantities of both being reported from stomachs I., Pugachev, O., Rusinek, O., Timoshkin, O. & Nascetti, G. (1995). Taxonomic rank and origin of Contracaecum of both C. grewingkii and C. inermis (Zubin 1992, osculatum baicalensis Mozgovoi and Ryjikov, 1950, parasite of Dzyuba et al. 2000, Dzyuba 2004). It is thus plausible Phoca sibirica from Lake Baikal, with data on its occurrence that C. osculatum baicalensis can be transmitted also in fish hosts. Abstracts of 4th International Symposium of by Baikal copepods (Rusinek 2007), as it is in the Fish Parasitology, Munich October 3-7, 27. case of marine copepods and C. osculatum s.l. (Koie Dogel’, V.A., Bogolepova, I.I. & Smirnova, K.V. (1949). & Fagerholm 1995). Should it be the case, it would Parazitofauna Bajkala i eë zoogeografičeskoe značenie, considerably increase the range of the nematode Vestnik Leningradskogo Universiteta 7: 13-34 (in Russian). targets, as the above-mentioned copepods are the Dzido, J. (2011). Identyfikacja molekularna oraz zróżnicowanie main food source of young C. grewingkii and C. wewnątrzgatunkowe pasożytniczych nicieni z rodziny inermis and thus the fish can be infected very early Anisakidae. Unpublished doctoral dissertation, University of by the larval C. osculatum baicalensis. As the above- Gdańsk, Gdańsk, Poland. Dzyuba, E.V. (2004). Issledowanie piŝčevyh strategii pelagičeskich mentioned fish species may be cannibalistic with ryb Bajkala. Unpublished doctoral dissertation, Institut respect to younger individuals (Zubin 1992, Dzyuba Biologii Vnutrennyh Vod im. I.D. Papanina RAN, Borok, et al. 2000, Dzyuba 2004), they may not only be the Russia (in Russian). second intermediate hosts, but also paratenic ones Dzyuba, E.V., Mel’nik, N.G. & Naumova, E.Y. (2000). Feeding on account of the consumed infected fish. spectra of the young of Cottocomephorus inermis (Cottidae) Valtonen et al. (1988) believe that the seal and in Lake Baikal. J. Ichthyol. 40: 342-345. their parasite populations from the Bothnian Bay Hoarau, G., Holla, S., Lescasse, R., Stam, W.T. & Olsen, J.L. and Lake Baikal have been isolated in the course of (2002). Heteroplasmy and evidence for recombination in the their evolution, which leads to the conclusion that mitochondrial control region of the flatfish Platichthys flesus. C. osculatum baicalensis is a separate species. It is Mol. Biol. Evol. 19(12): 2261–2264. also consistent with the results of this study where Kijewska, A., Rokicki, J., Sitko, J. & Węgrzyn, G. (2002). Ascaridoidea: A simple DNA assay for identification of www.oandhs.ocean.ug.edu.pl

©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 73 Contracaecum osculatum baicalensis from Baikal Cottocomephorus spp.

11 species infecting marine and freshwater fish, mammals, nerpy. Trudy Gel’mintologičeskoj Laboratorii 55: 59-66, (in and fish-eating birds. Exp. Parasitol. 101(1): 35–39. DOI: Russian). 10.1016/S0014-4894(02)00031-0. Valtonen, E.T., Fagerholm, H.P. & Helle, E. (1988). Koie, M. & Fagerholm, H. P. (1995). The life cycle of Contracaecum osculatum (Nematoda: Anisakidae) in fish Contracaecum osculatum (Rudolphi, 1802) sensu and seals from Bothnian Bay (Northeastern Baltic Sea). stricto (Nematoda, Ascaridoidea, Anisakidae) in view of Int. J. Parasitol. 18(3): 365-370. DOI: 10.1016/0020- experimental infections. Parasitol. Res. 81: 481-489. 7519(88)90146-4. Kozhov, M.M. (1963). Lake Baikal and its Life. Monogr. Biol. Zaika V.E. (1965). Parazitofauna ryb ozera Bajkal. Russia: 11, The Hague: Junk Publishers. Izdatel’stvo Nauka, (in Russian). Lâjman, E.M. (1933). Parazitičeskie červi ozera Bajkal. Trudy Zhu, X., D’Amelio, S., Paggi, L. & Gasser, R.B. (2000). Bajkalskoj Limnologičeskoj Stancji 4: 5-98 (in Russian). Assessing sequence variation in internal transcribed Margolis, L., Esch, G. W., Holmes, J. C., Kuris, A. M. & Schad spacers of ribosomal DNA within and among members G. A. (1982). The use of ecological terms in parasitology of the Contracaecum osculatum complex (Nematoda: (report of an ad hoc committee of the American Society of Ascaridoidea: Anisakidae). Parasitol. Res. 86(8): 677 – 683. Parasitologists), J. Parasitol. 68(1): 131 – 133. DOI: 10.1007/PL00008551. Mattiucci, S., Nascetti, G., Cianchi, R., Paggi, L., Arduino, P., Zhu, X., Gasser, R.B., Podolska, M. & Chilton, N.B. (1998). Margolis, L., Brattey, J., Webb, S., D’Amelio, S., Orecchia, Characterization of anisakid nematodes with zoonotic P. & Bullini, L. (1997). Genetic and ecological data on the potential by nuclear ribosomal DNA sequences. Int. J. Anisakis simplex complex, with evidence for a new species Parasitol. 28(12): 1911 – 1921. DOI: 10.1016/S0020- (Nematoda, Ascaridoidea, Anisakidae). J. Parasitol. 83(3): 7519(98)00150-7. 401-416. Zubin, A.A. (1992). Diet of benthopelagic Baikal Mattiucci, S. & Nascetti, G. (2007). Genetic diversity and (Scorpaeniformes, Cottoidei), J. Ichthyol. 32: 42-47. infection levels of anisakid nematodes parasitic in fish and marine mammals from Boreal and Austral hemispheres. Vet. Parasitol. 148: 43-57. DOI: 10.1016/j.vetpar.2007.05.009. Mattiucci, S. & Nascetti, G. (2008). Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host – parasite co – evolutionary processes. Adv. Parasitol. 66: 48 – 148. DOI: 10.1016/S0065-308X(08)00202-9. Mazepova, G.F. (1998). The role of copepods in the Baikal ecosystem. J. Mar. Syst. 15: 113–120. DOI: 10.1016/S0924- 7963(97)00065-1. Melnik, N.G., Timoshkin, O.A. & Sideleva, V.G. (1995). Distribution of M. branickii and some characteristics of its ecology. Guide and key to pelagic of Baikal. Novosibirsk: Izdatel’stvo Nauka. Rolbiecki, L. (2002). A rapid method for preparing semipermanent glycerol-jelly parasite Mount. Wiadomości Parazytologiczne 48: 87-88. Rudstam, L.G., Melnik, N.G., Timoshkin, O.A., Hansson, S., Pushkin, S.V. & Nemov, V. (1992). Diel Dynamics of an Aggregation of Macrohectopus Branickii (DYB.) (Amphipoda, Gammaridae) in the Barguzin Bay, Lake Baikal, Russia. J. Gt. Lakes Res. 18(2): 286-297. DOI 10.1016/S0380-1330(92)71296-9. Rusinek, O.T. (2007). Fish parasites of Lake Baikal (fauna, communities, zoogeography and historical background). Moscow: KMK Scientific Press Ltd. (in Russian). Sambrook, J., Fritsh, E.F. & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press. Sudarikov, V.E. & Ryzhikov, K.M. (1951). K biologii Contracaecum osculatum baicalensis – nematody bajkalskoj

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